Indoleamine 2,3-dioxygenase (IDO) catalyzes the oxidation of L-tryptophan to N-formyl kynurenine. In contrast to the wide spectrum of P450 monooxygenases, IDO is one of the only two heme-based dioxygenases in humans. Despite decades of effort, its dioxygenase mechanism remains elusive. IDO is an immunosuppressive enzyme, which plays an important role in allowing cancer cells to escape from immune surveillance. Recently, it has attracted a great deal of attention due to the recognition of its potential as a therapeutic target for cancer. Hence, there is a critical need for the delineation of the dioxygenase and inhibitory mechanisms of the two human isoforms of IDO, named hIDO1 and hIDO2. The long term goal of our program is (i) to define the molecular mechanism of heme-based dioxygenases, filling in the knowledge gap in heme oxygen chemistry, and (ii) to delineate the antitumor effect of IDO1/IDO2 inhibitors, aiding in the definition of IDO-linked cancer physiology. The objective of this application is to characterize the molecular properties of hIDO1 and hIDO2, in order to define their catalytic and inhibitory mechanisms. Our central hypothesis is that the catalytic and inhibitory mechanisms of the two IDO isoforms are distinct. The rationale is that the successful completion of this project will offer strong, conceptual and evidence-based guidelines for the development of IDO- targeted intervention against cancer. Thus, the proposed research is relevant to that part of NIH's mission that pertains to the development of fundamental knowledge that will potentially help to reduce the burdens of human disability. Guided by strong preliminary data, our hypothesis will be tested by the pursuit of two specific aims: (i) define the dioxygenase mechanisms of hIDO1 and hIDO2, and (ii) identify the inhibition mechanisms of hIDO1 and hIDO2. To achieve our objective we will employ a multi-faceted approach with a complementary set of spectroscopic techniques (Raman, UV-Vis, FTIR, EPR, MS and X-ray crystallography), combined with computational methodologies (MD and QM/MM) and mutagenesis. The proposed project is innovative because (i) the availability of both hIDO1 and hIDO2 places us in a unique position for carrying out the proposed comparative studies, and (ii) the unique fast-mixing/freeze-quenching techniques developed in our lab enable effective structural characterization of key enzymatic intermediates that are inaccessible in other labs. The proposed research is significant because the successful completion of this project will lay a solid foundation for the future design of novel therapeutic strategies targeting IDO, as well as to advance fundamental understanding of heme-based dioxygenase chemistry.